Gene expression profiling of homologous recombination repair pathway indicates susceptibility for olaparib treatment in malignant pleural mesothelioma in vitro.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a tumour arising from pleural cavities with poor prognosis. Multimodality treatment with pemetrexed combined with cisplatin shows unsatisfying response-rates of 40%. The reasons for the rather poor efficacy of chemotherapeutic treatment are largely unknown. However, it is conceivable that DNA repair mechanisms lead to an impaired therapy response. We hypothesize a major role of homologous recombination (HR) for genome stability and survival of this tumour. Therefore, we analysed genes compiled under the term "BRCAness". An inhibition of this pathway with olaparib might abrogate this effect and induce apoptosis. METHODS: We investigated the response of three MPM cell lines and lung fibroblasts serving as a control to treatment with pemetrexed, cisplatin and olaparib. Furthermore, we aimed to find possible correlations between response and gene expression patterns associated with BRCAness phenotype. Therefore, 91 clinical MPM samples were digitally screened for gene expression patterns of HR members. RESULTS: A BRCAness-dependent increase of apoptosis and senescence during olaparib-based treatment of BRCA-associated-protein 1 (BAP1)-mutated cell lines was observed. The gene expression pattern identified could be found in approx. 10% of patient samples. Against this background, patients could be grouped according to their defects in the HR system. Gene expression levels of Aurora Kinase A (AURKA), RAD50 as well as DNA damage-binding protein 2 (DDB2) could be identified as prognostic markers in MPM. CONCLUSIONS: Defects in HR compiled under the term BRCAness are a common event in MPM. The present data can lead to a better understanding of the underlaying cellular mechanisms and leave the door wide open for new therapeutic approaches for this severe disease with infaust prognosis. Response to Poly (ADP-ribose)-Polymerase (PARP)-Inhibition could be demonstrated in the BAP1-mutated NCI-H2452 cells, especially when combined with cisplatin. Thus, this combination therapy might be effective for up to 2/3 of patients, promising to enhance patients' clinical management and outcome.

Setting out the frame conditions for feasible use of FFPE derived RNA.

INTRODUCTION: The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis. DESIGN: FFPE-derived RNA from sections of 23 patients with spontaneous pneumothoraxes was used. Unstained sections of FFPE tissue were stored at various temperatures (-80 °C, -20 °C, 4 °C, 24 °C) prior to RNA extraction. The potential impact on RNA quality of semi-automatic and manual RNA isolation and three different deparaffinization agents (mineral oil, xylene and d-limonene) were compared. RESULTS: The storage temperature of FFPE sections affects RNA concentration and fragmentation, with the optimal storage temperature below -20 °C. The RNA extracted with d-limonene shows equivalent quality to the RNA extracted using more toxic standard agents. The manual isolation provides a higher RNA yield compared to the semi-automatic isolation. However, no differences in the amount of longer RNA fragments were observed. Furthermore, the semi-automatic isolation showed an enhanced RNA quality. CONCLUSION: FFPE sections not directly used for RNA extraction should be stored below -20 °C to increase quality and yield of the RNA. Usage of semi-automatic isolation produces superior results and simplifies routine processes by having less hands-on-time. Replacement of toxic xylene by d-limonene may contribute to improved occupational safety while not influencing analytical results.

Inhibition of MDM2 via Nutlin-3A: A Potential Therapeutic Approach for Pleural Mesotheliomas with MDM2-Induced Inactivation of Wild-Type P53.

Previously, our group demonstrated that nuclear expression of E3 ubiquitin ligase (MDM2) in malignant pleural mesothelioma (MPM) is significantly associated with decreased overall survival. A possible explanation may be that overexpression of MDM2 leads to a proteasomal degradation of TP53 that eventually results in a loss of TP53-induced apoptosis and senescence. It is well known from other tumor entities that restoration of TP53 activity, e.g., by MDM2 inhibition, results in an instant TP53-induced stress and/or DNA damage response of cancer cells. Nutlin-3A (a cis-imidazoline analogue) has been described as a potent and selective MDM2 inhibitor preventing MDM2-TP53-interaction by specific binding to the hydrophobic TP53-binding pocket of MDM2. In the present study, the effects of MDM2 inhibition in MPM via Nutlin-3A and standard platinum based chemotherapeutic agents were comparatively tested in three MPM cell lines (NCI-H2052, MSTO-211H, and NCI-H2452) showing different expression profiles of TP53, MDM2, and its physiological inhibitor of MDM2-P14/ARF. Our in vitro experiments on MPM cell lines revealed that Nutlin-3A in combination with cisplatin resulted in up to 9.75 times higher induction of senescence (p=0.0050) and up to 5 times higher apoptosis rate (p=0.0067) compared to the commonly applied cisplatin and pemetrexed regimens. Thus Nutlin-3A, a potent inhibitor of MDM2, is associated with a significant induction of senescence and apoptosis in MPM cell lines, making Nutlin-3A a promising substance for a targeted therapy in the subgroup of MPM showing MDM2 overexpression.

Immunohistochemically detectable metallothionein expression in malignant pleural mesotheliomas is strongly associated with early failure to platin-based chemotherapy.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a biologically highly aggressive tumor arising from the pleura with a dismal prognosis. Cisplatin is the drug of choice for the treatment of MPM, and carboplatin seems to have comparable efficacy. Nevertheless, cisplatin treatment results in a response rate of merely 14% and a median survival of less than seven months. Due to their role in many cellular processes, methallothioneins (MTs) have been widely studied in various cancers. The known heavy metal detoxifying effect of MT-I and MT-II may be the reason for heavy metal drug resistance of various cancers including MPM. METHODS: 105 patients were retrospectively analyzed immunohistochemically for their MT expression levels. Survival analysis was done by Cox-regression, and statistical significance determined using likelihood ratio, Wald test and Score (logrank) tests. RESULTS: Cox-regression analyses were done in a linear and logarithmic scale revealing a significant association between expression of MT and shortened overall survival (OS) in a linear (p=0.0009) and logarithmic scale (p=0.0003). Reduced progression free survival (PFS) was also observed for MT expressing tumors (linear: p=0.0134, log: p=0.0152). CONCLUSION: Since both, overall survival and progression-free survival are negatively correlated with detectable MT expression in MPM, our results indicate a possible resistance to platin-based chemotherapy associated with MT expression upregulation, found exclusively in progressive MPM samples. Initial cell culture studies suggest promoter DNA hypomethylation and expression of miRNA-566 a direct regulator of copper transporter SLC31A1 and a putative regulator of MT1A and MT2A gene expression, to be responsible for the drug resistance.

miRNA regulation is important for DNA damage repair and recognition in malignant pleural mesothelioma.

Platin-containing regimes are currently considered as state-of-the-art therapies in malignant pleural mesotheliomas (MPM) but show dissatisfying response rates ranging from 6 to 16% only. Still, the reasons for the rather poor efficacy remain largely unknown. A clear stratification of patients based on new biomarkers seems to be a promising approach to enhance clinical management, which would be a long-needed improvement for MPM patients but does not seem likely soon unless new biomarkers can be validated. Twenty-four formalin-fixed, paraffin-embedded (FFPE) tumour specimens were subjected to a miRNA expression screening of 800 important miRNAs using digital quantification via the nCounter technique (NanoString). We defined a small subset of miRNAs regulating the key enzymes involved in the repair of platin-associated DNA damage. Particularly, the TP53 pathway network for DNA damage recognition as well as genes related to the term "BRCAness" are the main miRNA targets within this context. The TP53 pathway network for DNA damage recognition as well as genes related to the term "BRCAness" are the main players for risk stratification in patients suffering from this severe disease. Taking the specific molecular profile of the tumour into account can help to enhance the clinical management prospectively and to smooth the way to better response prediction.

Massive parallel sequencing and digital gene expression analysis reveals potential mechanisms to overcome therapy resistance in pulmonary neuroendocrine tumors.

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. 25% show neuroendocrine differentiation (typical/atypical carcinoids, large-/small-cell neuroendocrine carcinomas). Carcinoids present with long survival rates, but metastatic carcinoids correlate with decreased survival and are commonly insensitive to standard chemotherapy or radiation. Therefore, novel therapeutic strategies are urgently needed. Material and methods: 70 representative tumor specimens were used for next-generation sequencing analysis of 14 genes related to therapy response. Additionally, mRNA-expression profiles of 60 matching samples were determined for 13 selected drug targets by using the NanoString nCounter technology. Results: A number of features known to sensitize tumors for different targeted therapies could be identified, which hopefully improve the clinical management of this subgroup of lung neoplasias. In particular, EGFR expression was observed in the investigated tumors in a noteworthy manner. Additionally, MDM2 was strongly expressed in the majority of all samples whereas the expression of its physiological inhibitor, CDKN2A, was nearly absent in all low-grade tumors. TP53 showed a high frequency of variants in high-grade tumors but mutations were rare in carcinoids. Conclusion: Based on our results, therapeutic approaches with MDM2-inhibitors and monoclonal anti-EGFR antibodies may be promising in pulmonary carcinoid tumors.

ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP Were Identified as Reference Genes in Neuroendocrine Lung Cancer via the nCounter Technology.

BACKGROUND: Neuroendocrine lung cancer (NELC) represents 25% of all lung cancer cases and large patient collectives exist as formalin-fixed, paraffin-embedded (FFPE) tissue only. FFPE is controversially discussed as source for molecular biological analyses and reference genes for NELC are poorly establishes. MATERIAL AND METHODS: Forty-three representative FFPE-specimens were used for mRNA expression analysis using the digital nCounter technology (NanoString). Based on recent literature, a total of 91 mRNA targets were investigated as potential tumor markers or reference genes. The geNorm, NormFinder algorithms and coefficient of correlation were used to identify the most stable reference genes. Statistical analysis was performed by using the R programming environment (version 3.1.1). RESULTS: RNA integrity (RIN) ranged from 1.8 to 2.6 and concentrations from 34 to 2,109 ng/µl. However, the nCounter technology gave evaluable results for all samples tested. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP were identified as constantly expressed genes with high stability (M-)values according to geNorm, NormFinder and coefficients of correlation. CONCLUSION: FFPE-derived mRNA is suitable for molecular biological investigations via the nCounter technology, although it is highly degraded. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP are potent reference genes in neuroendocrine tumors of the lung.

Screening of Pleural Mesotheliomas for DNA-damage Repair Players by Digital Gene Expression Analysis Can Enhance Clinical Management of Patients Receiving Platin-Based Chemotherapy.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons for the rather poor efficacy of platinum compounds remain largely unknown. MATERIAL AND METHODS: For this exploratory mRNA study, 24 FFPE tumour specimens were screened by digital gene expression analysis. Based on data from preliminary experiments and recent literature, a total of 366 mRNAs were investigated using a Custom CodeSet from NanoString. All statistical analyses were calculated with the R i386 statistical programming environment. RESULTS: CDC25A and PARP1 gene expression were correlated with lymph node spread, BRCA1 and TP73 expression levels with higher IMIG stage. NTHL1 and XRCC3 expression was associated with TNM stage. CHECK1 as well as XRCC2 expression levels were correlated with tumour progression in the overall cohort of patients. CDKN2A and MLH1 gene expression influenced overall survival in this collective. In the adjuvant treated cohort only, CDKN2A, CHEK1 as well as ERCC1 were significantly associated with overall survival. Furthermore, TP73 expression was associated with progression in this subgroup. CONCLUSION: DNA-damage response plays a crucial role in response to platin-based chemotherapeutic regimes. In particular, CHEK1, XRCC2 and TP73 are strongly associated with tumour progression. ERCC1, MLH1, CDKN2A and most promising CHEK1 are prognostic markers for OS in MPM. TP73, CDKN2A, CHEK1 and ERCC1 seem to be also predictive markers in adjuvant treated MPMs. After a prospective validation, these markers may improve clinical and pathological practice, finally leading to a patients' benefit by an enhanced clinical management.